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Measuring Procaspase-8 and -10 Processing upon Apoptosis Induction
细胞凋亡诱导产生半胱天冬酶原-8和半胱天冬酶原-10的测定   

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Abstract

Apoptosis or programmed cell death is important for multicellular organisms to keep cell homeostasis and for the clearance of mutated or infected cells. Apoptosis can be induced by intrinsic or extrinsic stimuli. The first event in extrinsic apoptosis is the formation of the Death-Inducing Signalling Complex (DISC), where the initiator caspases-8 and -10 are fully activated by several proteolytic cleavage steps and induce the caspase cascade leading to apoptotic cell death. Analysing the processing of procaspases-8 and -10 by Western blot is a commonly used method to study the induction of apoptosis by death receptor stimulation. To analyse procaspase-8 and -10 cleavage, cells are stimulated with a death ligand for different time intervals, lysed and subjected to Western blot analysis using anti-caspase-8 and anti-caspase-10 antibodies. This allows monitoring the caspase cleavage products and thereby induction of apoptosis.

Keywords: Caspase-8(半胱天冬酶-8), Cleavage(剪切), Proteolysis(蛋白水解), Western blot(蛋白质印迹), Apoptosis(细胞凋亡), Cell death(细胞死亡)

Background

Caspases are proteases that are produced as inactive zymogens and are activated by proteolytic cleavage (Degterev et al., 2003). The activation of the caspase cascade is the most important event during apoptotic cell death, which induces the typical biochemical and morphological changes of the apoptotic cell. In contrast to inactive executioner procaspases, the initiator procaspases 8/9/10 have restricted proteolytic activity and become fully activated in high molecular weight complexes (Lavrik et al., 2005). Procaspase-9 is activated at the platform termed apoptosome during intrinsic apoptosis, while stimulation of the death receptors TNF-R1 (Tumor Necrosis Factor Receptor 1), CD95/Fas (Cluster of Differentiation 95), TRAIL-R1 (Tumor Necrosis Factor Related Apoptosis Inducing Ligand Receptor 1), TRAIL-R2 (Tumor Necrosis Factor Related Apoptosis Inducing Ligand Receptor 2) leads to the recruitment of procaspases-8 and -10 with adapter proteins to form the Death-Inducing Signalling Complex (DISC) during extrinsic apoptosis (Schleich et al., 2012). Here, caspases-8 and -10 enter close proximity and perform several intra- and inter-molecular cleavages. This processing results in the release of a small and a large subunit from the prodomain. These form the active heterotetramers p182p102 and p172p122 that triggers the caspase cascade leading to the demolition of the cell (see Figures 1A and 1B).

To verify apoptosis induction, it is important to show the activation of caspases. Checking the activation of the initiator procaspases 8 and 10, accompanied by their cleavage, gives main evidence for the induction of the extrinsic apoptosis via death receptors. The kinetics of procaspases-8 and -10 processing can be analysed by monitoring its cleavage steps by Western blot (see Figure 1C) and give information about the induction of apoptotic cell death after death receptor stimulation (Schleich et al., 2016). Depending on the part of the caspase that is recognized by the antibody, the intermediate products of the caspase containing this particular part can be analysed by western blot. Healthy, unstimulated cells only contain the unprocessed procaspases-8 and -10 (p55/53 and p59/55, respectively), but after stimulation the amount of procaspase decreases and the intermediate cleavage products (caspase-8 p43/41 or caspase-10 p47/43) and active subunits (caspase-8 p18) are enriched and can be detected by Western blot (Figure 1C). By performing time-dependent analysis, it is possible to follow the course of caspase cleavage including the enrichment of the cleaved forms (Schleich et al., 2016). This also allows to compare the time-dependent cleavage of caspases between several conditions.

To ensure getting the complete information on procaspase-8/-10 processing we have developed the protocol presented below. In contrast to a number of other protocols for Western blot analysis of procaspase-8/-10 processing, here we do not follow only a part of the Western blot of a specific molecular weight, e.g., by cutting the membrane, or use antibodies specific only to the active forms of the caspases. In this way, we can follow simultaneously several cleavage products of procaspases-8 and -10: proform, intermediate and final cleavage products. Only this way of measuring procaspase-8/-10 processing allows to escape from misjudgement on the efficiency of procaspase cleavage, e.g., in some studies only the proform of procaspases is followed and in case of a low stimulation strength and a weak caspase processing, the small differences in the decrease of the proform are not detected and might be misleading. Furthermore, another very important feature of our protocol is performing the measurement over different time intervals, which is also often neglected, and thus, the results might be misleading due to missing the key time points of processing, for example the appearance of active caspases, that are degraded fast. To the later point, there is a cell type and stimulation strength specificity with respect to the timing of procaspase-8/-10 processing and the corresponding time intervals have to be carefully selected in each particular case.


Figure 1. Kinetics of procaspase-8 and procaspase-10 processing. A. Scheme of caspase-8 processing. The cleavage at D216, D374 and D384 results in the release of the active subunits (p18 and p10). The point marks the region of the caspase that is recognized by the antibody (epitope). B. Scheme of caspase-10 processing. The cleavage at D219, D372 results in the release of the active subunits (p23/p17 and p12). The point marks the region of the caspase that is recognized by the antibody (epitope). C. Western blot: Hela-CD95 cells (Neumann et al., 2010) were stimulated with 250 ng/ml CD95L for the indicated periods of time. Samples were lysed, subjected to SDS-PAGE and analysed by Western blot for caspase-8 and caspase-10. Western blot for actin was used as loading control.

Materials and Reagents

  1. 6-well plates (with a surface suitable for your cells, e.g., SARSTEDT, catalog number: 83.3920 )
  2. Microcentrifuge tubes (e.g., VWR, catalog number: 89202.684 )
  3. Cuvettes (e.g., SARSTEDT, model: 67.742 )
  4. Blotting membrane and Whatman paper (e.g., Trans-Blot Turbo Transfer Pack Mini incl. 0.2 µm nitrocellulose and transfer buffer, Bio-Rad Laboratories, catalog number: 170-4158 )
  5. Human cells expressing caspase-8 and caspase-10, here: HeLa cells stably overexpressing CD95 (Neumann et al., 2010; DKMZ, catalog number: ACC 57 )
  6. Suitable medium for your cells (e.g., DMEM/Ham’s F12 including 10% fetal bovine serum and 1% penicillin/streptomycin)
  7. CD95L has been prepared as described in Fricker et al., 2010
  8. Bovine serum albumin (BSA) standard 2 mg/ml (Bio-Rad Laboratories, catalog number: 5000260 )
  9. SDS-poly acrylamide gel (e.g., Mini-PROTEAN TGX stain-free precast gels, 12%, Bio-Rad Laboratories, catalog number: 456-8046 )
  10. Protein assay dye reagent concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
  11. Protein standard (e.g., Precision plus Protein All Blue standards, Bio-Rad Laboratories, catalog number: 1610373 )
  12. Anti-caspase-8 antibody, clone C15 (final concentration 0.5-2 µg/ml, a kind gift of P. Krammer, DKFZ Heidelberg)
  13. Anti-caspase-10 antibody, clone 4C1 (dilution 1:1,000, final concentration 1 µg/ml, MEDICAL & BIOLOGICAL LABORATORIES, catalog number: MBL-M059-3 )
  14. Anti-actin antibody (dilution: 1:4,000, final concentration 0.125-0.2 µg/ml, Sigma-Aldrich, catalog number: A2103 )
  15. HRP-coupled isotype specific secondary antibodies (e.g., Santa Cruz Biotechnology, catalog numbers: sc-2060 , sc-2004 , sc-2062 )
  16. HRP-Substrate for Enhanced Luminescence, (e.g., Luminata Forte Western HRP Substrate, EMD Millipore, catalog number: WBLUF0500 )
  17. 10x PBS (Biochrom, catalog number: L1835 )
  18. cOmpleteTM protease inhibitor cocktail (PIC) (Roche Diagnostics, catalog number: 11 836 145 001 )
  19. Tris (AppliChem, catalog number: A2264 )
  20. Natrium chloride (NaCl) (Carl Roth, catalog number: P029.3 )
  21. EDTA (Carl Roth, catalog number: CN06.2 )
  22. Glycerol (Carl Roth, catalog number: 3783.1 )
  23. Triton (Carl Roth, catalog number: 3051.4 )
  24. Tween-20 (AppliChem, catalog number: A1389 )
  25. Milk powder (Carl Roth, catalog number: T145.5 )
  26. Sodium azide (Carl Roth, catalog number: K305.1 )
  27. β-mercaptoethanol (Carl Roth, catalog number: 4227.2 ).
  28. 10 x Tris/Glycine/SDS (Bio-Rad Laboratories, catalog number: 161-0723 )
  29. 4x Laemmli sample buffer (Bio-Rad Laboratories, catalog number: 161-0747 )
  30. 1x PBS (see Recipes)
  31. Protease inhibitor cocktail (PIC, see Recipes)
  32. Lysis buffer (see Recipes)
  33. 1 x Tris/Glycine/SDS (see Recipes)
  34. PBS-T (see Recipes)
  35. Blocking buffer (see Recipes)
  36. Primary antibody dilution (see Recipes)
  37. 4x Laemmli sample buffer (reducing, see Recipes)

Equipment

  1. CO2-incubator (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: BBD 6220 )
  2. Cell scraper (e.g., Orange Scientific, catalog number: 4460600N )
  3. Centrifuge (Eppendorf, model: 5418R )
  4. Photometer (Bio-Rad Laboratories, model: SmartSpec Plus Spectrophotometer )
  5. Electrophoresis chamber and power supply (Bio-Rad Laboratories, model: Mini-PROTEAN Tetra cell and PowerPac HC )
  6. Transfer system (Bio-Rad Laboratories, model: Trans-Blot Turbo Transfer System )
  7. Imager (Bio-Rad Laboratories, model: ChemiDoc XRS+ Imaging System )

Software

  1. Image Lab (Bio-Rad Laboratories)

Procedure

  1. Cell lysis
    1. Plate 2.5 x 105 HeLa-cells per well on 6-wells in 2 ml medium (see Materials and Reagents 6) and incubate overnight at 37 °C, 5% CO2.
    2. Next day control cells, aspirate medium and replace with fresh medium. Stimulate cell with apoptosis inducing agent (here 250 ng/ml CD95L) in a time-dependent manner (here: 20, 40, 60, 90, 120 min). Stimulation dose and time is cell line dependent and have to be tested for each cell line. Stimulation should be performed in a way that one 6-well plate can be harvested at once.
    3. Incubate the cells at 37 °C, 5% CO2.
    4. Add cold PBS to the cells (do not aspirate media), harvest them by scraping and transfer medium including cells into a new tube (everything on ice). Be careful to transfer all the cells into the new tube.
    5. Centrifuge at 4 °C, 500 x g for 5 min.
    6. Discard supernatant, but not the cell pellet!
    7. Add 1 ml of cold PBS.
    8. Centrifuge at 4 °C, 500 x g for 5 min.
    9. Aspirate and discard supernatant.
    10. Store the pellet at -20 °C or continue with cell lysis by resuspending the cell pellet directly in 30 µl ice-cold lysis buffer (see Recipe 3), be careful to resuspend it completely.
    11. Incubate on ice for 30 min, vortex 2-3 times.
    12. Centrifuge at 4 °C, 16,400 x g for 15 min.
    13. Transfer the supernatant (= lysate) into a new tube, discard cell pellet. The supernatant should have a clear, yellowish colour.

  2. Measurement of protein concentration
    1. Measure protein concentration by mixing 2 µl of lysate with 1,000 µl Bio-Rad assay dye reagent (see Recipe 4) in a cuvette.
    2. Prepare a BSA-standard with dilutions according to manufacturer’s instructions.
    3. Mix 2 µl lysis buffer with 1,000 µl Bio-Rad solution and use it as blank.
    4. Incubate at room temperature for 5 min. The blank should have a brown-red colour, while the samples with lysates should turn into blue.
    5. Measure protein concentration for samples and BSA-standard at 595 nm with Bio-Rad SmartSpec plus or other spectrophotometer. Follow instructions of spectrophotometer for protein standard. Be careful to measure within the linear range and to dilute samples with too high protein concentrations. The expected protein concentration is between 3 and 5 mg/ml.

  3. SDS-PAGE and blot transfer
    1. Prepare 25 µg of protein lysate with lysis buffer and 4x Laemmli sample buffer (reducing, see Recipe 9) to a final volume of 15 µl (e.g., mix 5 µl of a 5 µg/µl lysate with 6.25 µl lysis buffer and 3.75 µl sample buffer). If the protein concentration is too low you can use gels with higher loading volume or decrease protein amount to 20 µg.
    2. Heat the samples at 95 °C for 5 min.
    3. Spin down shortly to avoid steam on the lid.
    4. Prepare a chamber with gel and 1x Tris/Glycine/SDS (Sodium dodecyl sulfate) buffer (see Recipe 5).
    5. Load samples on 12% Bio-Rad pre-cast stain-free gels. Be careful to load the complete sample into the well without leakage and spillover. Use of 25 µl Hamilton syringe or long pipette tips makes loading on a gel easier. Put the end of the tip or the syringe to the bottom of the well and very slowly push the sample into the well. Then carefully remove the Hamilton syringe or the tip from the well without spillover of the sample. When using Hamilton syringe rinse it three times in-between loading the next sample.
    6. Load 2 µl of all blue protein standard.
    7. Run the gel with 150 V until the protein dye passes the bottom of the gel. Check the progress from time to time.
    8. Place gel in Tris/Glycine/SDS buffer.
    9. If you use Bio-Rad stain-free gels, activate the dye in the gel with one minute UV light, take a picture of the gel and check the separation of the proteins. It is not necessary to use Bio-Rad stain-free gel system. The system is based on the implementation of chemical compounds that react with tryptophan under UV light resulting in fluorescence. The fluorophore is visible on the membrane after blotting.
    10. Prepare a sandwich consisting of Whatman paper sheets, a membrane (PVDF or Nitrocellulose) = Blot, the gel and Whatman paper sheets in presence of a transfer buffer. Be careful to place the sandwich in the blotting system oriented to the cathode, so that the proteins will be transferred from the gel to the membrane, not into the Whatman paper. Blot with Transblot Turbo transfer system according to instruction with 2.5 A, 25 V for 9 min. It is possible to use a different system for Western blot as well.
    11. Rinse blot with PBS-T (PBS-Tween, see Recipe 6) for a short time.
    12. If you are using Bio-Rad stain-free gels, take a picture of the blot and check blotting results for air bubbles and equal transfer.

  4. Membrane blocking and detection of caspase-10
    1. Incubate in blocking buffer (see Recipe 7) for 1 h shaking at room temperature. The blot should be covered completely with buffer.
    2. Rinse 3 x for 5 min with PBS-T, spill buffer into the dish, not directly onto the blot to avoid removing of proteins.
    3. Incubate with primary antibody dilution (see Recipe 8) overnight shaking at 4 °C. You can use the antibody solution several times, but store it a 4 °C and check for contamination before using it. When the antibody signal is fading, prepare a new antibody solution.
    4. Wash blot 4 x with PBS-T for 5 min, shaking at room temperature.
    5. Prepare a 1:10,000 dilution of secondary antibody in blocking buffer, approx. 20 ml/blot.
    6. Incubate the blot in the secondary antibody dilution for 60 min at room temperature while shaking.
    7. Wash blot 3 x with PBS-T for 10 min while shaking at room temperature.
    8. Rinse blot with 1 ml Luminata forte solution and wait a few seconds, the complete blot should be covered with the solution.
    9. Place blot in the imager machine and scan the blot.
    10. Wash blot 3 x in PBS-T for 5 min.

  5. Detection of caspase-8 and actin
    1. Repeat steps of D3-D10 for caspase-8 and actin antibody detection. The use of isotype specific secondary antibodies allows to detect caspase-8 after caspase-10 on the same membrane. Alternatively it is possible to use two separate blots for detection of caspase-8 and 10 (see Figure 1C).

Data analysis

Check the quality of your blot, the bands should appear sharp without any smear or air bubbles. By adding a positive control to your gel, it will be possible to check the quality of the blot and the detection of the caspase fragments. For example, the caspase-8 cleavage products p43/41 and p18 should be visible (Figure 1C). A possible positive control could be lysate from apoptotic dying cells (e.g., HeLa-CD95 cells stimulated for 120 min with 250 ng/ml CD95L). Next to this qualitative analysis of the Western blot data, they can be quantified, too. This requires a high-quality blot and antibody signals within the detection range (no overexposure). There are several approaches to perform the quantitative analysis, one can be found here: Schleich et al., 2016.

Notes

  1. Mice do not express caspase-10, they only express caspase-8.
  2. The optimal time point and concentration for detection of caspase-8 and -10 activation have to be optimized for each cell line and stimulus.
  3. You can use the primary antibody dilution several times until it is contaminated or efficiency of detection is fading. Store at 4 °C.
  4. It is possible to use TBS-T instead of PBS-T for blot washing.
  5. Washing buffers can be prepared with deionized water instead of MilliQ water.

Recipes

  1. 1x PBS
    For 1 L add 100 ml 10x PBS to 900 ml MilliQ water
  2. Protease inhibitor cocktail (PIC)
    1 tablet of cOmpleteTM protease inhibitor cocktail
    Dilute in 2 ml of MilliQ water to receive 25x PIC solution
    Store in aliquots at -20 °C
  3. Lysis buffer (store at 4 °C)
    20 mM Tris (pH 7.4)
    137 mM NaCl
    2 mM EDTA
    10% glycerol
    1% Triton
    Add 4% (v/v) 25x PIC at the day of use (see Recipe 2)
  4. Bio-Rad protein assay dye reagent (100 ml)
    For 100 ml add 20 ml Bio-Rad protein assay dye reagent concentrate to 80 ml MilliQ water
    Use up to 3 weeks and store at RT
  5. 1x Tris/glycine/SDS
    For 1 L add 100 ml 10 x Tris/glycine/SDS to 900 ml water
  6. PBS-T
    Add 2 ml Tween-20 to 2 L 1x PBS and mix
  7. Blocking buffer
    Mix 50 g milk powder in 1 L PBS-T until solved completely
  8. Primary antibody dilution
    Dilute antibody according to manufacturer’s instruction (see Materials and Reagents) in PBS-T Add sodium azide to a final concentration of 0.1% (w/v)
    Store the antibody dilution at 4 °C
  9. 4x Laemmli sample buffer (reducing)
    Mix 900 µl of 4x Laemmli sample buffer with 100 µl β-mercaptoethanol according to the manufacturer’s instructions

Acknowledgments

The work has been supported by RSF 14-44-00011. This protocol was adapted from Schmidt et al., 2015; Pietkiewicz et al., 2015 and Schleich et al., 2016.

References

  1. Degterev, A., Boyce, M. and Yuan, J. (2003). A decade of caspases. Oncogene 22(53): 8543-8567.
  2. Fricker, N., Beaudouin, J., Richter, P., Eils, R., Krammer, P. H. and Lavrik, I. N. (2010). Model based dissection of CD95 signaling dynamics reveals both a pro- and anti-apoptotic role of c-FLIPL. J Cell Biol 190(3): 377-389.
  3. Lavrik, I. N., Golks, A. and Krammer, P. H. (2005). Caspases: pharmacological manipulation of cell death. J Clin Invest 115(10): 2665-2672.
  4. Neumann, L., Pforr, C., Beaudouin, J., Pappa, A., Fricker, N., Krammer, P. H., Lavrik, I. N. and Eils, R. (2010). Dynamics within the CD95 death-inducing signaling complex decide life and death of cells. Mol Syst Biol 6(1): 352.
  5. Pietkiewicz, S., Eils, R., Krammer, P. H., Giese, N. and Lavrik, I. N. (2015). Combinatorial treatment of CD95L and gemcitabine in pancreatic cancer cells induces apoptotic and RIP1-mediated necroptotic cell death network. Exp Cell Res 339(1): 1-9.
  6. Schleich, K., Buchbinder, J. H., Pietkiewicz, S., Kahne, T., Warnken, U., Ozturk, S., Schnolzer, M., Naumann, M., Krammer, P. H. and Lavrik, I. N. (2016). Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain. Cell Death Differ 23(4): 681-694
  7. Schleich, K., Warnken, U., Fricker, N., Ozturk, S., Richter, P., Kammerer, K., Schnolzer, M., Krammer, P. H. and Lavrik, I. N. (2012). Stoichiometry of the CD95 death-inducing signaling complex: experimental and modeling evidence for a death effector domain chain model. Mol Cell 47(2): 306-319.
  8. Schmidt, J. H., Pietkiewicz, S., Naumann, M. and Lavrik, I. N. (2015). Quantification of CD95-induced apoptosis and NF-κB activation at the single cell level. J Immunol Methods 423: 12-7.

简介

细胞凋亡或程序性细胞死亡对于多细胞生物保持细胞稳态和清除突变或感染细胞是重要的。 细胞凋亡可以由内在或外在的刺激诱导。 外源性凋亡中的第一个事件是死亡诱导信号复合物(DISC)的形成,其中引发剂半胱天冬酶-8和-10通过若干蛋白水解切割步骤完全活化并诱导胱天蛋白酶级联导致凋亡细胞死亡。 通过蛋白质印迹分析procaspases-8和-10的处理是通过死亡受体刺激研究凋亡诱导的常用方法。 为了分析procaspase-8和-10切割,用死亡配体刺激细胞不同的时间间隔,裂解,并使用抗半胱天冬酶-8和抗半胱天冬酶-10抗体进行蛋白质印迹分析。 这可以监测胱天蛋白酶切割产物,从而诱导细胞凋亡。

背景 胱天蛋白酶是作为无活性酶原产生并被蛋白水解切割活化的蛋白酶(Degterev等人,2003)。胱天蛋白酶级联的激活是凋亡细胞死亡期间最重要的事件,其诱导凋亡细胞的典型生化和形态学变化。与非活性执行者procaspases相反,启动子胱天蛋白酶8/9/10具有限制性蛋白水解活性,并在高分子量复合物中完全活化(Lavrik等人,2005)。促凋亡受体TNF-R1(肿瘤坏死因子受体1),CD95 / Fas(分化群95),TRAIL-R1(肿瘤坏死因子相关细胞凋亡)诱导配体受体1),TRAIL-R2(肿瘤坏死因子相关凋亡诱导配体受体2)导致在外源性细胞凋亡期间募集胱天蛋白酶-8和-10与衔接蛋白形成死亡诱导信号复合物(DISC)(Schleich 等人,2012)。在这里,胱天蛋白酶-8和-10密切相邻并进行几个分子间和分子间切割。这种处理导致从前域释放一个小的和大的亚基。这些形成了触发半胱天冬酶级联的活性异源四聚体p18 p10 2和p17 p12 2 拆除电池(见图1A和1B)。
 为了验证凋亡诱导,重要的是显示胱天蛋白酶的激活。检查启动子胱天蛋白酶8和10的激活伴随其切割,为通过死亡受体诱导外源性凋亡提供了主要证据。可以通过用蛋白质印迹法(见图1C)监测其切割步骤并分析死亡受体刺激后凋亡细胞死亡诱导的信息,分析procaspases-8和-10处理的动力学(Schleich等, em>,2016)。取决于被抗体识别的半胱天冬酶的部分,可以通过蛋白质印迹分析含有该特定部分的半胱天冬酶的中间产物。健康的未刺激的细胞只含有未经加工的胱天蛋白酶-8和-10(分别为p55 / 53和p59 / 55),但刺激后,胱天蛋白酶的量减少,中间裂解产物(半胱天冬酶-8 p43 / 41或半胱天冬酶-10 p47 / 43)和活性亚单位(半胱天冬酶-8 p18)富集,可通过Western印迹检测(图1C)。通过进行时间依赖性分析,可以跟踪胱天蛋白酶切割的过程,包括裂解形式的富集(Schleich等人,2016)。这也允许比较几种条件下胱天蛋白酶的时间依赖性切割。
 为了确保获得有关procaspase-8 / -10处理的完整信息,我们制定了以下协议。与用于procaspase-8 / -10加工的Western印迹分析的许多其它方案相反,在这里,我们不仅仅通过切割仅遵循特定分子量的蛋白质印迹的一部分,例如
膜,或使用仅针对胱天蛋白酶的活性形式特异的抗体。这样,我们可以同时遵循胱天蛋白酶-8和-10的几种切割产物:前体,中间和最终的裂解产物。只有这种测量procaspase-8 / -10处理的方式才能避免对过敏原酶切割效率的错误判断,例如,在一些研究中,仅遵循胱天蛋白酶的形式,并且在低刺激的情况下强度和弱胱天蛋白酶处理,形态的减少的微小差异没有被检测到,可能是误导性的。此外,我们的协议的另一个非常重要的特征是在不同的时间间隔执行测量,这也经常被忽略,因此,由于缺少处理的关键时间点,例如活动半胱氨酸蛋白酶的出现,结果可能是误导性的,那是快速降解的。对于晚期,相对于procaspase-8 / -10处理的定时,存在细胞类型和刺激强度特异性,并且在每个特定情况下必须仔细选择相应的时间间隔。


图1. procaspase-8和procaspase-10加工的动力学。A.半胱天冬酶-8处理方案。 D216,D374和D384处的切割导致活性亚单位的释放(p18和p10)。 该点表示由抗体(表位)识别的胱天蛋白酶的区域。 B.胱天蛋白酶-10处理方案。 D219,D372的切割导致活性亚单位的释放(p23 / p17和p12)。 该点表示由抗体(表位)识别的胱天蛋白酶的区域。 C. Western印迹:用250ng / ml CD95L刺激Hela-CD95细胞(Neumann et al。,2010)指定的时间段。 将样品裂解,进行SDS-PAGE,并通过Western印迹分析胱天蛋白酶-8和半胱天冬酶-10。 蛋白质印迹用作肌动蛋白作为加载对照。

关键字:半胱天冬酶-8, 剪切, 蛋白水解, 蛋白质印迹, 细胞凋亡, 细胞死亡

材料和试剂

  1. 6孔板(适合您的细胞的表面,例如,,SARSTEDT,目录号:83.3920)
  2. 微量离心管(例如,VWR,目录号:89202.684)
  3. 比色皿(例如, SARSTEDT,型号:67.742)
  4. 印迹膜和Whatman纸(例如,Trans-Blot Turbo Transfer Pack Mini包含0.2μm硝酸纤维素和转移缓冲液,Bio-Rad Laboratories,目录号:170-4158)
  5. 表达半胱天冬酶-8和半胱天冬酶-10的人细胞,这里:稳定过表达CD95的HeLa细胞(Neumann等人,2010; DKMZ,目录号:ACC 57)
  6. 适用于您的细胞的培养基(例如,包含10%胎牛血清和1%青霉素/链霉素的DMEM / Ham's F12)
  7. CD95L已经按照Fricker等人,2010所述进行了制备
  8. 牛血清白蛋白(BSA)标准2mg / ml(Bio-Rad Laboratories,目录号:5000260)
  9. SDS-poly丙烯酰胺凝胶(例如,Mini-PROTEAN TGX无染色预制凝胶,12%,Bio-Rad Laboratories,目录号:456-8046)
  10. 蛋白测定染料试剂浓缩物(Bio-Rad Laboratories,目录号:500-0006)
  11. 蛋白质标准品(例如,精密加蛋白全蓝标准品,Bio-Rad Laboratories,目录号:1610373)
  12. 抗半胱氨酸蛋白酶-8抗体,克隆C15(终浓度为0.5-2微克/毫升,一种亲切的礼物P.Krammer,DKFZ海德堡)
  13. 抗半胱天冬酶-10抗体,克隆4C1(稀释度1:1,000,终浓度1μg/ ml,MEDICAL& BIOLOGICAL LABORATORIES,目录号:MBL-M059-3)
  14. 抗肌动蛋白抗体(稀释度:1:4,000,终浓度0.125-0.2μg/ ml,Sigma-Aldrich,目录号:A2103)
  15. HRP偶联同种型特异性第二抗体(例如,Santa Cruz Biotechnology,目录号:sc-2060,sc-2004,sc-2062)
  16. 用于增强发光的HRP-底物(例如,Luminata Forte Western HRP底物,EMD Millipore,目录号:WBLUF0500)
  17. 10倍PBS(Biochrom,目录号:L1835)
  18. cOmplete TM蛋白酶抑制剂混合物(PIC)(Roche Diagnostics,目录号:11 836 145 001)
  19. Tris(AppliChem,目录号:A2264)
  20. 氯化钠(NaCl)(Carl Roth,目录号:P029.3)
  21. EDTA(Carl Roth,目录号:CN06.2)
  22. 甘油(Carl Roth,目录号:3783.1)
  23. Triton(Carl Roth,目录号:3051.4)
  24. Tween-20(AppliChem,目录号:A1389)
  25. 奶粉(Carl Roth,目录号:T145.5)
  26. 叠氮化钠(Carl Roth,目录号:K305.1)
  27. β-巯基乙醇(Carl Roth,目录号:4227.2)。
  28. 10×Tris / Glycine / SDS(Bio-Rad Laboratories,目录号:161-0723)
  29. 4x Laemmli样品缓冲液(Bio-Rad Laboratories,目录号:161-0747)
  30. 1x PBS(参见食谱)
  31. 蛋白酶抑制剂鸡尾酒(PIC,参见食谱)
  32. 裂解缓冲液(见配方)
  33. 1×Tris / Glycine / SDS(参见食谱)
  34. PBS-T(参见食谱)
  35. 阻塞缓冲区(见配方)
  36. 初次抗体稀释(参见食谱)
  37. 4x Laemmli样品缓冲液(减少,见配方)

设备

  1. CO 2,例如Thermo Fisher Scientific,Thermo Scientific,型号:BBD 6220)的培养基(例如,
  2. 细胞刮刀(例如,,Orange Scientific,目录号:4460600N)
  3. 离心机(Eppendorf,型号:5418R)
  4. 光度计(Bio-Rad Laboratories,型号:SmartSpec Plus分光光度计)
  5. 电泳室和电源(Bio-Rad Laboratories,型号:Mini-PROTEAN Tetra电池和PowerPac HC)
  6. 转运系统(Bio-Rad Laboratories,型号:Trans-Blot Turbo Transfer System)
  7. 成像仪(Bio-Rad Laboratories,型号:ChemiDoc XRS +成像系统)

软件

  1. 图像实验室(Bio-Rad Laboratories)

程序

  1. 细胞裂解
    1. 在2ml培养基中的6孔的孔中每孔2.5×10 5 HeLa细胞(参见材料和试剂6),并在37℃,5%CO 2 / sub>。
    2. 第二天对照细胞,吸出培养基并用新鲜培养基代替。以时间依赖性方式(这里:20,40,60,90,120分钟)刺激具有凋亡诱导剂(这里为250ng / ml CD95L)的细胞。刺激剂量和时间是细胞系依赖性的,必须对每个细胞系进行测试。刺激应以一个6孔板可以一次收获的方式进行。
    3. 在37℃,5%CO 2孵育细胞。
    4. 向细胞中加入冷的PBS(不要吸入培养基),通过刮擦并将包含细胞的培养基转移到新管(冰上的一切)中收获它们。小心将所有细胞转移到新管中。
    5. 在4℃,500×g离心5分钟。
    6. 丢弃上清液,而不是细胞沉淀!
    7. 加入1 ml冷PBS。
    8. 在4℃,500×g离心5分钟。
    9. 吸出并弃去上清液。
    10. 将沉淀物保存在-20°C或通过将细胞沉淀物直接重悬在30μl冰冷的裂解缓冲液中(见方案3),继续细胞裂解,注意将其完全重新悬浮。
    11. 在冰上孵育30分钟,旋转2-3次。
    12. 在4℃,16,400×g离心15分钟。
    13. 将上清液(=裂解物)转移到新管中,弃去细胞沉淀。上清液应具有清晰,淡黄色的颜色
  2. 测量 的蛋白质浓度
    1. 通过在比色皿中混合2μl裂解物和1,000μlBio-Rad测定染料试剂(参见方案4)来测量蛋白质浓度。
    2. 根据制造商的说明准备BSA标准稀释液。
    3. 混合2μl裂解缓冲液与1,000μlBio-Rad溶液,并将其用作空白。
    4. 在室温下孵育5分钟。空白应为棕红色,而含裂解物的样品应变成蓝色。
    5. 用Bio-Rad SmartSpec plus或其他分光光度计测量595 nm样品的蛋白质浓度和BSA标准品。按照分光光度计的指示进行蛋白质标准。注意在线性范围内测量,并以高蛋白浓度稀释样品。预期蛋白质浓度在3至5 mg / ml之间。

  3. SDS-PAGE和印迹转移
    1. 使用裂解缓冲液和4x Laemmli样品缓冲液(还原,见方法9)准备25μg蛋白质裂解物至终体积为15μl(例如,将5μl5μg/μl裂解物与6.25 μl裂解缓冲液和3.75μl样品缓冲液)。如果蛋白质浓度太低,您可以使用更高负荷量的凝胶或将蛋白质量减少至20μg。
    2. 将样品在95℃加热5分钟。
    3. 短时间旋转以避免盖上的蒸汽。
    4. 用凝胶和1×Tris / Glycine / SDS(十二烷基硫酸钠)缓冲液制备室(参见方案5)。
    5. 在12%Bio-Rad预浸渍无色凝胶上载样。小心将完整的样品装入井中,不会泄漏并溢出。使用25μlHamilton注射器或长移液管吸头可以使凝胶更容易上胶。将尖端或注射器的末端放在井的底部,并非常缓慢地将样品推入井中。然后小心地从孔中取出Hamilton注射器或尖端,而不会溢出样品。当使用汉密尔顿注射器时,将三次加载下一个样品。
    6. 加载2μl所有蓝色蛋白质标准品。
    7. 用150 V运行凝胶,直到蛋白质染料通过凝胶底部。检查不时的进度。
    8. 将凝胶置于Tris / Glycine / SDS缓冲液中。
    9. 如果使用Bio-Rad无染色凝胶,用一分钟的紫外线激活凝胶中的染料,拍摄凝胶图像并检查蛋白质的分离。不需要使用Bio-Rad无染色凝胶体系。该系统基于在紫外光下与色氨酸反应产生荧光的化合物的实施。印迹后荧光团在膜上是可见的。
    10. 在转移缓冲液存在下准备由Whatman纸片,膜(PVDF或硝化纤维素)=印迹,凝胶和Whatman纸张组成的三明治。小心将三明治置于面向阴极的印迹系统中,使蛋白质从凝胶转移到膜上,而不是进入Whatman论文。按照2.5 A,25 V,9分钟的说明,用Transblot Turbo传输系统打印。也可以使用不同的系统进行蛋白质印迹。
    11. 用PBS-T(PBS-Tween,见方案6)冲洗印迹短时间。
    12. 如果您使用Bio-Rad无染色凝胶,请拍摄印迹,并检查吸气结果是否有气泡和相等的转移。

  4. 半胱天冬酶-10的膜阻断和检测
    1. 在封闭缓冲液(见方案7)中孵育1小时,室温振荡。印迹应完全用缓冲液覆盖。
    2. 用PBS-T冲洗3×5分钟,将溢出缓冲液冲洗至培养皿中,不直接冲洗到印迹上,以避免蛋白质的去除。
    3. 孵育与一次抗体稀释(见配方8)在4℃过夜振荡。您可以多次使用抗体溶液,但将其储存在4°C,并在使用前检查是否有污染。当抗体信号褪色时,制备新的抗体溶液。
    4. 用PBS-T清洗4×5分钟,在室温下振荡。
    5. 在阻断缓冲液中准备1:10,000稀释的二抗,约20毫升/印迹。
    6. 在室温下振荡时,在二次抗体稀释液中孵育60分钟。
    7. 在室温下振荡的同时用PBS-T清洗3×10分钟。
    8. 用1ml Luminata forte溶液冲洗印迹并等待几秒钟,完全印迹应覆盖溶液。
    9. 在成像机中放置印迹并扫描印迹。
    10. 在PBS-T中3×洗涤5分钟。

  5. 检测caspase-8和肌动蛋白
    1. 重复步骤D3-D10为caspase-8和肌动蛋白抗体检测。使用同种型特异性第二抗体可以在同一膜上检测半胱天冬酶-10后的半胱天冬酶-8。或者,可以使用两个单独的印迹来检测半胱天冬酶-8和10(参见图1C)。

数据分析

检查您的印迹的质量,乐队应该看起来没有任何涂片或气泡。通过向凝胶中添加阳性对照,可以检查印迹的质量和半胱天冬酶片段的检测。例如,caspase-8切割产物p43 / 41和p18应该是可见的(图1C)。可能的阳性对照可能是来自凋亡性死亡细胞(例如,用250ng / ml CD95L刺激120分钟的HeLa-CD95细胞)的裂解物。除了Western印迹数据的定性分析之外,它们也可以量化。这需要在检测范围内的高质量印迹和抗体信号(无过度曝光)。执行定量分析有几种方法可以在这里找到:Schleich等人, 2016。

笔记

  1. 小鼠不表达半胱天冬酶-10,它们只表达半胱天冬酶-8。
  2. 用于检测半胱天冬酶-8和-10活化的最佳时间点和浓度必须针对每种细胞系和刺激进行优化。
  3. 您可以多次使用一次抗体稀释,直到被污染或检测效率正在下降。储存于4°C。
  4. 可以使用TBS-T代替PBS-T进行污点清洗。
  5. 洗涤缓冲液可用去离子水代替MilliQ水制备。

食谱

  1. 1x PBS
    对于1升,加入100毫升10倍的PBS至900毫升MilliQ水
  2. 蛋白酶抑制剂鸡尾酒(PIC)
    1片cOmplete TM 蛋白酶抑制剂混合物
    稀释于2ml MilliQ水中,以接收25x PIC溶液 储存于-20°C等分试样
  3. 裂解缓冲液(4℃储存)
    20mM Tris(pH7.4)
    137 mM NaCl
    2 mM EDTA
    10%甘油
    1%Triton
    在使用当天添加4%(v / v)25x PIC(见配方2)
  4. Bio-Rad蛋白测定染料试剂(100ml)
    对于100 ml加入20ml Bio-Rad蛋白质测定染料试剂浓缩至80 ml MilliQ水中 使用长达3周,并在RT存储
  5. 1×Tris /甘氨酸/ SDS 对于1L,将100ml 10×Tris /甘氨酸/ SDS加入到900ml水中
  6. PBS-T
    加入2ml Tween-20至2L 1x PBS并混合
  7. 阻塞缓冲区
    将50g奶粉在1L PBS-T中混合,直到完全解决
  8. 初级抗体稀释度
    根据制造商的说明(见材料和试剂)在PBS-T中稀释抗体添加叠氮化钠至最终浓度为0.1%(w / v)
    将抗体稀释液储存在4°C
  9. 4x Laemmli样品缓冲液(还原)
    根据制造商的说明书
    将900μl4x Laemmli样品缓冲液与100μlβ-巯基乙醇混合

致谢

这项工作得到了RSF 14-44-00011的支持。该协议由Schmidt等人,2015年改编; Pietkiewicz等人,2015年和Schleich等人,2016年。

参考文献

  1. Degterev,A.,Boyce,M.and Yuan,J.(2003)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/14634618"目标="_ blank">十年的胱天蛋白酶。癌基因 22(53):8543-8567。
  2. Fricker,N.,Beaudouin,J.,Richter,P.,Eils,R.,Krammer,PH和Lavrik,IN(2010)。< a class ="ke-insertfile"href ="https:// www .ncbi.nlm.nih.gov / pubmed / 20696707"target ="_ blank"> CD95信号传导动力学基于模型的解剖揭示了c-FLIPL的促凋亡作用和抗凋亡作用。 Biol。190(3):377-389。
  3. Lavrik,IN,Golks,A.and Krammer,PH(2005)。  胱天蛋白酶:细胞死亡的药理作用。 J Clin Invest 115(10):2665-2672。
  4. Neumann,L.,Pforr,C.,Beaudouin,J.,Pappa,A.,Fricker,N.,Krammer,PH,Lavrik,IN和Eils,R。(2010)。< a class = insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/?term=Dynamics+within+the+CD95+death-inducing+signaling+complex+decide+life+and+death+of+细胞。"目标="_ blank"> CD95死亡诱导信号复合物内的动力学决定了细胞的生命和死亡。"Mol Syst Biol"6(1):352.
  5. Pietkiewicz,S.,Eils,R.,Krammer,PH,Giese,N。和Lavrik,IN(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm。 nih.gov/pubmed/26453936"target ="_ blank">胰腺癌细胞中CD95L和吉西他滨的组合治疗诱导凋亡和RIP1介导的坏死细胞死亡网络。 Exp Cell Res 339 (1):1-9。
  6. Schleich,K.,Buchbinder,JH,Pietkiewicz,S.,Kahne,T.,Warnken,U.,Ozturk,S.,Schnolzer,M.,Naumann,M.,Krammer,PH和Lavrik,IN(2016)。   DISC中DED链的分子结构:通过短DED蛋白c-FLIP和procaspase-8前域的胱天蛋白酶原-8活化。细胞死亡差异23(4):681-694
  7. Schleich,K.,Warnken,U.,Fricker,N.,Ozturk,S.,Richter,P.,Kammerer,K.,Schnolzer,M.,Krammer,PH和Lavrik,IN(2012)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22683265"target ="_ blank"> CD95死亡诱导信号复合物的化学计量学:实验和建模证据死亡效应域结构域模型。分子细胞 47(2):306-319。
  8. Schmidt,JH,Pietkiewicz,S.,Naumann,M.and Lavrik,IN(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed / 25967949"target ="_ blank">在单细胞水平上CD95诱导的细胞凋亡和NF-κB活化的定量.JISA免疫方法423:12-7。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Pietkiewicz, S., Wolfe, C., Buchbinder, J. H. and Lavrik, I. N. (2017). Measuring Procaspase-8 and -10 Processing upon Apoptosis Induction. Bio-protocol 7(1): e2081. DOI: 10.21769/BioProtoc.2081.
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